Autoimmunity Reviews 12 (2013) 543–553
`
`Contents lists available at SciVerse ScienceDirect
`
`Autoimmunity Reviews
`
`j o u r n a l h om e p a g e : ww w . e l s e v i e r . c o m / l o c a t e / a u t r e v
`
`Review
`The mechanism of action of glatiramer acetate in multiple sclerosis and beyond
`Rina Aharoni ⁎
`Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100, Israel
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 2 September 2012
`Accepted 19 September 2012
`Available online 7 October 2012
`
`Keywords:
`Multiple sclerosis (MS)
`Experimental autoimmune encephalomyelitis
`(EAE)
`Glatiramer acetate (GA)
`Immunomodulation
`Neuroprotection
`Autoimmune related pathologies
`
`In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), the
`immune system reacts again self myelin constitutes in the central nervous system (CNS), initiating a detri-
`mental inflammatory cascade that leads to demyelination as well as axonal and neuronal pathology. The
`amino acid copolymer glatiramer acetate (GA, Copaxone) is an approved first-line treatment for MS that
`has a unique mode of action. Accumulated evidence from EAE-induced animals and from MS patients indi-
`cates that GA affects various levels of the innate and the adaptive immune response, generating deviation
`from the pro-inflammatory to the anti-inflammatory pathway. This review aims to provide a comprehensive
`perspective on the diverse mechanism of action of GA in EAE/MS, in particular on the in situ immunomodu-
`latory effect of GA and its ability to generate neuroprotective repair consequences in the CNS. In view of its
`immunomodulatory activity, the beneficial effect of GA in various models of other autoimmune related
`pathologies, such as immune rejection and inflammatory bowel disease (IBD) is noteworthy.
`© 2012 Elsevier B.V. All rights reserved.
`
`Contents
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`1.
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`4.
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`5.
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`Introduction — the pathology of multiple sclerosis and the development of glatiramer acetate .
`Peripheral immunomodulatory mechanisms
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`Immunomodulation in the CNS .
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`Neuroprotection and repair processes .
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`4.1.
`Elevation of neurotrophic factors
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`4.2.
`Reduced CNS injury .
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`4.3. Myelin repair and neurogenesis
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`The effect of GA in additional autoimmune related pathologies .
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`5.1.
`Transplantation systems of graft versus host disease and graft rejection
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`5.2.
`Stem cell transplantation .
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`5.3.
`Inflammatory bowel disease .
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`Conclusions .
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`6.
`Take-home messages .
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`References .
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`Abbreviations: MS, Multiple sclerosis; EAE, Experimental autoimmune encephalomy-
`elitis; CNS, Central nervous system; BBB, Blood brain barrier; MBP, Myelin basic protein;
`MOG, Myelin oligodendrocyte glycoprotein; PLP, Myelin proteolipid protein; GA,
`Glatiramer acetate; APC, Antigen presenting cells; MHC, Major histocompatibility; TCR,
`T-cell receptor; APL, Altered peptide ligand; Th, T-helper; Tregs, T-regulatory cells;
`Foxp3, Forkhead box P3; IL, Interleukin; IFN, interferon; TGF, Transforming growth
`factor; NT, Neurotrophin; BDNF, Brain derived neurotrophic factor; IGF, Insulin-like
`growth factor; MRI, Magnetic resonance imaging; MTR, Magnetization transfer ratio;
`DTI, Diffusion tensor imaging; SEM, Scanning electron microscopy; TEM, Transmission
`electron microscopy; OPCs, Oligodendrocyte progenitor cells; NPCs, Neuronal progenitor
`cells GVHD, Graft versus host disease; IBD, Inflammatory bowel disease; CD, Crohn's
`disease.
`⁎ Tel.: +972 8 9342997; fax: +972 8 9344141.
`E-mail address: rina.aharoni@weizmann.ac.il.
`
`1568-9972/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
`http://dx.doi.org/10.1016/j.autrev.2012.09.005
`
`1. Introduction — the pathology of multiple sclerosis and the
`development of glatiramer acetate
`
`Multiple sclerosis (MS) is a chronic inflammatory demyelinating
`disease of the central nervous system (CNS) and a leading cause for
`disability in young adults, with female predominance [1]. The most
`typical clinical progression pattern is a phase of relapsing and remit-
`ting symptoms (relapsing remitting MS, RRMS) that frequently de-
`velops to a progressive disease course (secondary progressive MS,
`SPMS). A fraction of patients shows disease progression from the be-
`ginning (primary progressive MS, PPMS) which represents a somewhat
`different pathology [2]. Essential data on MS has been obtained by using
`
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`the animal model - experimental autoimmune encephalomyelitis
`(EAE), induced by immunization with myelin antigens such as myelin
`basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) or
`myelin proteolipid protein (PLP). EAE induction by the different en-
`cephalitogenic antigens or their peptides in susceptible animal strains
`leads to development of various disease forms (acute, relapsing remit-
`ting or progressive) that mimic the different patterns of MS. In spite of
`certain discrepancies, the EAE model has been proven as an essential
`tool for testing novel therapies as well as for the elucidation of their
`mechanism of action [3].
`Traditionally MS has been considered an autoimmune disease, in
`which the immune system reacts against the body's own constitu-
`ents, in this case against the myelin in the CNS, initiating a vicious in-
`flammatory cascade [4,5]. Several myelin encephalitogenic epitopes
`were identified in MS and EAE, such as the peptides comprising the
`84–102 amino acids of MBP, the 35–55 amino acids of MOG, and the
`139–151 amino acids of PLP [6]. In addition, epitope spreading toward
`multiple encephalitogenic determinants occurs with disease progres-
`sion [7]. Immune cells of both the adaptive and innate systems are
`involved in the inflammatory network that mediates the disease [8,9].
`T-helper (Th)-1 and Th-17 cells, cytotoxic T-cells, B-cells and macro-
`phages enter the CNS through the blood brain barrier (BBB) and the
`
`plexus choroideus, secreting pro-inflammatory cytokines, chemokines
`and other inflammatory substances. CNS resident cells, such as microg-
`lia and astrocytes, are stimulated upon tissue damage and further facil-
`itate T-cells activation and scar formation. All these cell populations
`maintain the inflammatory milieu and mediate tissue injury, leading
`to multifocal demyelination, impairment of nerve fiber conductivity,
`as well as loss of axons, neurons and oligodendrocytes (illustrated in
`Fig. 1). Defective T-cell apoptosis also plays a role in the development
`of the disease and its chronic evolution [10]. Besides the detrimental
`role of the immune system in MS/EAE pathology, immune cells such
`as Th2/3 and T-regulatory cells mediate anti-inflammatory protective
`pathways that suppress the disease. During the recent years it has be-
`come clear that MS is a multifaceted heterogeneous disease with differ-
`ent patterns of tissue damage [2]. Thus, in addition to the detrimental
`inflammation, widespread axonal and neuronal pathology is a central
`component of MS [9,11]. There is evidence that axonal transection and
`neuronal damage, occur even at early disease stage, supporting neuro-
`degenerative disease course [12]. Diffuse abnormalities in the grey
`matter and in normal-appearing brain tissue are currently recognized
`as central components of MS [13].
`Until about 20 years ago, only symptomatic treatments were avail-
`able for MS patients.
`Indiscriminate immunosuppression with its
`
`Fig. 1. Immune mediated pathological and modulatory pathways in MS and EAE.
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`hazardous risks and severe side effects was also attempted in order to
`restrain the excessively active immune system [14]. The development
`of the first disease-modifying therapies (DMTs) and their approval in
`the 1990s has altered the natural history of the disease. These are the re-
`combinant versions of interferon (IFN)-β [15,16] and the synthetic co-
`polymer glatiramer acetate (GA). The later is the subject matter of this
`review.
`In the late 1960s, Sela, Arnon, Teitelbaum and colleagues at the
`Weizmann Institute in Israel, conducting basic research on the immu-
`nological properties of synthetic polymers and copolymers, made a
`serendipitous drug discovery [17]. Hypothesizing that synthetic poly-
`peptides with amino acids analogous to those of the autoantigen MBP
`will induce EAE, they designed several such copolymeric mixtures.
`However, none of the copolymers was encephalitogenic, instead,
`they were found to be protective against EAE. Copolymer 1 (Cop 1),
`now called glatiramer acetate (GA; Copaxone), randomly composed
`of L-alanine, L-lysine, L-glutamic acid and L-tyrosine, in a molar ratio
`of 4.2:3.4:1.4:1.0, proved to have the greatest activity in ameliorating
`EAE. Furthermore, the effect of GA was not restricted to a particular
`species, disease type, or encephalitogen used for EAE induction, and
`it ameliorated disease in guinea pigs, rabbits, various mouse strains,
`and in two kinds of monkeys [18]. Since this discovery, extensive preclin-
`ical research, pivotal controlled clinical trials, and long-term assessments,
`established the efficacy and the safety of GA as a disease-modifying
`therapy for MS [19–22]. GA at a daily subcutaneous dose of 20 mg has
`been found to alter the natural history of RRMS by reducing the relapse
`rate and affecting disability. These consequences were consistent with
`magnetic resonance imaging (MRI) findings from various clinical centers
`[23]. Currently, GA is one of the most widely prescribed first-line treat-
`ments for RRMS.
`GA is the first and so far the only therapeutic agent ever to have a
`copolymer as its active ingredient. Due to the complexity and vari-
`ability of its polypeptides mixture, a clear definition of its active com-
`ponent has not been established. Moreover, it has been claimed that
`the overall “pool” of various amino acid sequences facilitates multiple
`ways of action and is thus required for its therapeutic activity. In
`some cases diverse findings led to contrasting conclusions and con-
`troversies as to which is the “key” process mediated by this drug.
`Due to these unique characteristics, GA has been often referred to as
`a drug with an “unclear” or “elusive” mechanism of action. Neverthe-
`less, over the last 4 decades, results from ours as well as from other
`laboratories, obtained in multiple in vitro and in vivo systems, clarified
`the immunological mechanism of action of GA (Table 1). Furthermore,
`our recent studies revealed neuroprotective repair consequences of GA
`treatment in the CNS. The purpose of this review is to provide a compre-
`hensive overview and elucidate the current understanding on the diverse
`mechanism of action of GA in EAE/MS. In addition findings on the effect
`of GA in additional autoimmune related pathologies are presented.
`
`2. Peripheral immunomodulatory mechanisms
`
`The immunological mechanism by which GA induces its therapeu-
`tic effect was extensively investigated over the years in EAE-induced
`animals and in MS patients. These studies indicated that GA acts by
`immunomodulating various levels of the immune response, which
`differ in their degree of specificity.
`The initial prerequisite step is the binding of GA to major histocom-
`patibility (MHC) class II molecules. In vitro studies on murine and
`human antigen presenting cells (APCs) indicated that GA undergoes a
`rapid and efficient (“promiscuous”) binding to various MHC class II
`molecules, and even displaces other peptides from the MHC binding
`groove [24]. This competition for binding to the histocompatibility mol-
`ecules can prevent the presentation of other antigens and hinder their
`T-cell activation. Several groups have demonstrated that GA induces
`generalized alterations of various types of APCs, such as dendritic cells
`
`and monocytes, so that they preferentially stimulate protective anti-
`inflammatory responses. Hence, dendritic cells from GA-treated MS
`patients produced less TNF-α, less IL-12, and more IL-10, compared to
`those of untreated patients [25]. GA induced a broad inhibitory effect
`on monocyte reactivity [26], and promoted the development of
`anti-inflammatory type II monocytes characterized by increased secre-
`tion of IL-10 and TGF-β, as well as by decreased production of IL-12 and
`TNF [27]. Furthermore, GA-induced type II monocytes were able to
`transfer protection from EAE [27]. This modulation on the level of the
`innate immune system is the least specific step in the immunological
`processes affected by GA, and can be beneficial for the inhibition of
`response to several myelin antigens. In addition, in the case of the
`MS immunodominant encephalitogenic epitope of MBP (comprising
`amino acids 82–100), GA acts in a strictly antigen-specific manner.
`Using MBP 82–100 specific T-cell clones from MS patients and from
`EAE-induced mice, it was shown that GA inhibits their activation by
`T-cell receptor (TCR) antagonism, acting as an altered peptide ligand
`(APL) [28].
`Most studies attribute the primary mechanism for GA activity to its
`ability to skew T-cell response from the pro-inflammatory to the
`anti-inflammatory pathway. It has been long known that GA-treated
`animals develop specific T cells in their peripheral immune system
`[18]. Furthermore, T-cell lines and hybridomas could be isolated from
`spleens of mice rendered unresponsive to EAE by GA [29]. Both cell
`types acted as modulatory suppressor cells, as they inhibited the re-
`sponse of MBP-specific effector cells in vitro, and adoptively transferred
`protection against EAE in vivo. T-cell lines/clones induced by GA pro-
`gressively polarized toward the T-helper (Th) 2/3 subtype, secreting
`high amounts of anti-inflammatory cytokines such as interleukin (IL)
`-4, -5, -10, and transforming growth factor (TGF)-β, until they com-
`pletely lost the ability to secrete Th1 pro-inflammatory cytokines such
`as INF-γ [30]. In several cases, the secretion of Th2/3 cytokines by the
`GA-induced T-cell lines was obtained in response to either GA or MBP.
`Other myelin antigens such as PLP and MOG could not activate the
`GA-specific T-cells, yet EAE induced by PLP and MOG could be
`suppressed by GA as well as by GA-induced T-cells. These results are in-
`dicative of “bystander suppression mechanisms” induced by GA [31]
`which are especially important in view of the epitope spreading occur-
`ring in MS/EAE [6,7]. A shift from a pro-inflammatory Th1-biased cyto-
`kine profile toward anti-inflammatory Th2-biased profile was observed
`also in GA-treated MS patients [32,33], indicating that such GA-specific
`cells are involved in the therapeutic effect of this drug in MS.
`The effect of GA on the T-cell subset is not restricted to the Th2/3
`versus Th1 pathways. Several studies demonstrated the effect of GA
`on Th-17 and on T-regulatory (Tregs) cells, which are pivotal effectors
`of disease exacerbation and suppression, respectively [34]. Thus, it
`was shown that in vitro exposure of peripheral CD4+ T-cells, from
`healthy humans or from GA-immunized mice, to GA, resulted in ele-
`vated level of Tregs, through activation of the transcription factor
`forkhead box P3 (Foxp3). Furthermore, GA treatment led to increased
`Foxp3 expression in CD4+ T-cells of MS patients, whose Foxp3 level
`was low at baseline [35]. Pretreatment of mice with GA, before EAE
`induction, resulted in increased Foxp3 expression on Tregs during
`the mild disease which was developed subsequently. These Tregs
`were more effective in EAE prevention than Tregs isolated from
`untreated mice [36]. GA treatment to EAE-induced mice resulted in
`elevation of Tregs and reduction of Th-17 cells, as demonstrated by
`the detection of their specific transcription factors, Foxp3 and RORγt,
`respectively, on both the mRNA and the protein levels [37,38]. In addi-
`tion to its effect on the CD4+ T-cell subset, GA affects also CD8+ T-cells.
`The regulatory function of these cells which was impaired in MS
`untreated patients, was drastically improved after several month of
`GA treatment, to the levels observed in healthy individuals [39,40].
`B-cells are involved in both the pathogenesis and modulation of
`MS and EAE, by secreting antibodies and cytokines as well as by
`their efficient antigen presentation [41]. GA treated patients
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`Table 1
`Immunomodulatory and neuroprotective effects of glatiramer acetate in MS and EAE
`
`Competition for MHC
`
`Promiscuous binding to various MHC class II molecules,
`
`Alteration of the innate immune response
`
`displacement of myelin antigens from the MHC binding groove [24].
`
`Inhibitory effect on monocytes reactivity, deviation of dendritic cells and
`
`monocytes to produce less TNF- and IL-12, more IL-10 and TGF- , and to
`
`stimulate Th2 anti-inflammatory responses [25–27].
`
`T-cell receptor antagonism
`
`Inhibition of the activation of T-cells specific to the 82-100 epitope of MBP [28].
`
`Induction of specific Th2/3-cells that secrete high amounts of IL-4, IL-5, IL-10, and TGF- [29–34].
`
`Elevation of the prevalence and function of T-regulatory cells, activation of the
`
`T-cell deviation
`
`transcription factor Foxp3 [35,36].
`
`Reduction of Th-17 cells and their transcription factors ROR t [37,38].
`Improvement of the regulatory function of CD8+ T-cells [39,40].
`
`Modification of B-cells
`
`Bias toward production of anti-inflammatory cytokines such as IL-10 [43].
`
`Induction of antibodies with beneficial rather than neutralizing activity [42].
`
`Down-regulating of chemokine receptors [44].
`
`GA-specific Th2/3 cells cross the BBB and secrete in situ
`
`anti-inflammatory cytokines.
`
`Secretion of anti-inflammatory cytokine
`
`Bystander expression of IL-10 and TGF- by resident
`
`astrocyte and microglia.
`
`Reduction in the overall expression of IFN- [46,47,49,50].
`
`Th-17 and T-regulatory cells
`
`Decrease in the amount of Th-17 cells. Increase in T-regulatory cells [38].
`
`Elevation of neurotrophic factors
`
`Restoration of the impaired expressions of BDNF, NT-3,
`
`GA-specific T-cells express BDNF in the brain [49].
`
`NT4, IGF-1, and IGF-2 [53–55,59].
`
`Prevention of demyelination [60–62]. Preservation of retinal
`
`ganglion cells [63]. Inhibition of motor neuron loss [62].
`
`Reduced CNS injury
`
`Preservation of brain tissue integrity by the MRI parameters
`
`MTR and DTI [65]. Reduced formation of “black holes” [69].
`
`Remyelination
`
`Neurogenesis
`
`Increase in NAA:Cr ratio [70].
`
`Augmented remyelination [62].
`
`Increased proliferation, maturation and
`
`survival of oligodendrocyte progenitor
`
`cells and their accumulation in the
`
`lesions [55,61].
`
`Elevated proliferation, migration and
`
`differentiation of neuronal progenitor
`
`cells and their recruitment into injury
`
`sites [64].
`
`Peripheral immunomodulation
`
`Immunomodulation in the CNS
`
`Neuroprotection
`
`Inserts demonstrate in situ consequences of GA in the CNS: A. GA-specific T-cells (blue) expressing IL-10 (red); B. infiltration of Foxp3 expressing T-cells (yellow); C. GA-specific
`T-cells (blue) expressing BDNF (red); D. intact formation of motor neurons; E. oligodendrocyte progenitor cells (red) extending processes between transected fibers (green);
`F. remyelination zone of newly myelinated axons surrounding an oligodendrocyte; G. BrdU expressing neuronal progenitors (yellow) born during GA/BrdU injection in a lesion
`site; BrdU expressing neuron (yellow) born during GA/BrdU injection, expressing mature neuronal marker in the cortex (green).
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`developed GA-specific antibodies that did not interfere with GA activ-
`ity in terms of MHC binding or T-cell stimulation, and eventually de-
`clined 6 month after treatment initiation [42]. The high proportion of
`IgG1 versus IgG2 antibody isotype as well as the switch to IgG4
`observed in the treated patients reflected the Th1 to Th2 shift induced
`by GA. Moreover relapse-free patients developed higher GA-antibody
`titers, suggesting a beneficial rather than neutralizing activity of
`anti-GA antibodies. Recently, it has been demonstrated that the effect
`of GA on B cells contributes to its therapeutic activity, leading to their
`biases toward the production of anti-inflammatory cytokines such as
`IL-10 [43]. These B cells ameliorated EAE by down-regulation of che-
`mokine receptors associated with trafficking of inflammatory cells
`into the CNS [44].
`The above cumulative findings from many laboratories established
`the broad immunomodulatory effect of GA on various subsets of the
`immune system.
`
`cerebrospinal fluid (CSF) of GA-treated MS patients revealed pro-
`nounced anti-inflammatory profile [50]. These cumulative results indi-
`cate that GA induces a bystander immunomodulatory effect in the CNS
`and generates in situ pro-inflammatory to anti-inflammatory cytokine
`shift, thus restraining the immuno-pathological disease progression.
`However, the effect of GA is not restricted to anti-inflammation, as de-
`scribed in the following.
`
`4. Neuroprotection and repair processes
`
`An essential challenge for MS therapy is to target not only the in-
`flammatory aspect of the disease but also its neuroaxonal pathology,
`aiming toward neuroprotective outcomes. By broad definition, neuro-
`protection is an effect that results in salvage, recovery, or regeneration
`of the nervous system, its cells, structure and function. During the re-
`cent years accumulated findings indicated that GA treatment generates
`neuroprotective consequences in the CNS.
`
`3. Immunomodulation in the CNS
`
`4.1. Elevation of neurotrophic factors
`
`The significant outcome of a therapy is obviously its effect in the
`diseased organ – in the case of MS – the ability to induce effective
`modulation of the pathological processes in the CNS. The initial im-
`munological activity of GA apparently occurs in the periphery (at
`the injection sites and in the corresponding draining lymph nodes).
`An indication for dendritic uptake of GA and its delivery to the CNS
`has been demonstrated [45]. However, since GA is rapidly degraded
`in the periphery, it is unlikely that its sufficient amounts can reach
`to the CNS to compete effectively with myelin antigens or initiate
`specific immune response. Most views thus currently accept that
`the therapeutic effect of GA is mediated by the GA-induced immune
`cells that penetrate the CNS. The presence in the CNS of GA-specific
`T-cells, induced in the periphery either by parenteral or by oral treat-
`ment, was demonstrated by their actual isolation from the brains of
`actively sensitized mice, as well as by their localization in the brain
`following passive transfer to the periphery [46,47]. Thus, specific
`ex-vivo reactivity to GA, manifested by cell proliferation and by Th2
`cytokine secretion, was found in whole lymphocyte population isolat-
`ed from brains of EAE induced mice treated by GA. Moreover, highly
`reactive GA-specific T-cell
`lines, that secreted in vitro IL-4, IL-5,
`IL-10 and TGF-β in response to GA, and cross-reacted with MBP at
`the level of Th2 cytokine secretion, were obtained from brains and
`spinal cords of GA-treated mice. The ability of the GA-induced cells
`to cross the blood–brain barrier (BBB) and accumulate in the CNS
`was confirmed by the injection of labeled GA-specific T-cells into
`the periphery and their subsequent detection in the brain [46,47].
`Preferential recruitment of GA-induced T-cells into inflamed organs,
`namely the brain in the case of EAE and the intestine in the case of in-
`flammatory bowel disease, was also demonstrated (Fig. 2). There is
`currently a consensus that the brain is not an immune privileged
`site and that activated T cells, regardless of their specificity, penetrate
`the CNS, especially in the course of MS/EAE when the BBB is dispirited
`[48]. While cross-reactivity of the GA-specific T-cells with MBP
`[29,30] is not essential for the entrance into the CNS, it may enable
`their in situ re-activation.
`In the CNS of EAE-induced mice, GA-specific T-cells manifested in-
`tense expression of the two potent regulatory anti-inflammatory cyto-
`kines IL-10 and TGF-β, but no trace of the detrimental inflammatory
`cytokine IFN-γ [49]. Of special interest is the finding that IL-10 and
`TGF-β were expressed not only by the GA-specific T-cells but also by
`CNS resident cells in their vicinity, such as astrocytes and microglia. In
`contrast, the overall expression of IFN-γ in the brain tissue was drasti-
`cally reduced. In addition, GA treatment resulted in drastic reduction
`in the occurrence of the pro-inflammatory Th-17 cells, with parallel
`elevation of Tregs in the CNS of mice with either chronic or relapsing–
`remitting EAE [38]. Analysis of GA-reactive T-cells
`from the
`
`The initial indication for neuroprotective activity was the ability of
`GA-induced cells to secrete not only anti-inflammatory cytokines, but
`also the potent brain derived neurotrophic factor BDNF. This was
`demonstrated for murine GA-specific T-cells originating from the pe-
`riphery or the CNS, as well as for human T-cell lines [51–54]. Further-
`more, GA-specific T-cells demonstrated extensive BDNF expression in
`the brain of EAE-induced mice [49]. In addition to the GA specific
`T-cells that penetrated the CNS, most of the BDNF positive cells
`were neurons and astrocytes that showed higher BDNF expression
`
`Fig. 2. Preferential recruitment of GA-induced T-cells into the inflamed organ, detec-
`tion by in vivo imaging system (IVIS). TDIR-labeled GA specific cells were adoptively
`transferred to mice inflicted with EAE (MOG-induced model), or with inflammatory
`bowel disease (dextran-induced model), or to naïve mice. IVIS imaging depicts brains,
`intestine and spleens from naïve, EAE and IBD mice, 10 days after cells transfer.
`
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`R. Aharoni / Autoimmunity Reviews 12 (2013) 543–553
`
`than in untreated EAE mice, thus confirming the bystander effect of
`GA on the CNS resident cells. BDNF was elevated also in brains of
`mice that were injected daily (subcutaneously) with GA as such, paral-
`lel to the practice used in the treatment of MS patients [54]. A similar
`phenomenon was found in brains of GA-treated mice for additional
`neurotrophic factors such as neurotrophin (NT)-3 and NT4 [54], as
`well as for insulin-like growth factor (IGF)-1 [55] and IGF-2 [56].
`Members of the neurotrophin family such as BDNF, NT-3 and NT-4
`are important regulators of neuronal function and survival [57]. Be-
`sides their well-established role in neuronal development, they have
`the capacity to promote axonal outgrowth, remyelination and regener-
`ation [57,58]. The elevation of these factors in the diseased organ may
`thus be of functional relevance and promote neuroprotective conse-
`quences. Of special significance is the finding that GA elevated the
`neurotrophic factors levels even when treatment started late - in the
`chronic disease phase, when they were drastically deteriorated. Re-
`duced levels of BDNF in the serum and the cerebral spinal fluid of MS
`patients, and its reversal by GA treatment, have been also reported
`[59], suggestive of the relevance of this effect to human therapy.
`
`4.2. Reduced CNS injury
`
`The neuroprotective effect of GA was manifested by actual preser-
`vation of the CNS and reduction in the typical EAE/MS tissue damage.
`Several studies, that utilized immunohistochemistry or electron
`microscopy, demonstrated protective outcome of GA on the primary
`target of the EAE/MS pathological process — the myelin [60–62]. Fur-
`thermore, in animals inflicted by MOG induced-EAE, in which chronic
`disease progression with extensive neurodegeration are typically
`manifested, GA treatment resulted in less neuroaxonal damage. This
`was evident by preservation of retinal ganglion cells [63], less axonal
`deterioration and fewer deformed neurons [64]. Motor neuron loss
`that occurs in this model was also prevented by GA treatment [62].
`In a recent study that employed advanced MRI parameters such as
`magnetization transfer ratio (MTR) and diffusion tensor imaging
`(DTI) for assessment of the whole brain, as well as for detection of
`specific affected regions, GA restored all the MRI parameters in both
`chronic and relapsing–remitting EAE models [65]. These finding indi-
`cate higher brain tissue integrity following GA treatment.
`The above beneficial effects were obtained by various regimens
`of GA administration. When treatment was applied before the ap-
`pearance of clinical manifestations, thus blocking the development
`of the pathological processes (prevention regimen), mice displayed
`nearly no damage. Moreover, when treatment was applied by a thera-
`peutic schedule, after disease exacerbation (suppression regimen) or
`even late in the chronic phase, when substantial injury was already
`manifested (delayed suppression regimen), significant reduction in my-
`elin and neuroaxonal damages was obtained. This suggested the induc-
`tion of genuine repair mechanisms.
`
`4.3. Myelin repair and neurogenesis
`
`The central elements of the CNS – the myelinating oligodendro-
`cytes as well as the neurons – are terminally differentiated cells
`with a limited capacity to respond to injury. They depend for renewal
`on the availability of their precursors — the oligodendrocyte progen-
`itor cells (OPCs) and the neuronal progenitor cells (NPCs), which
`need to undergo proliferation, migration and differentiation into the
`defined progeny. It should be noted, that CNS injury as such triggers
`repair processes [66]. In MS and EAE, subsequent to the demyelina-
`tion and degeneration, the opposing neuroprotective mechanisms —
`remyelination and neurogenesis are stimulated and progenitor cells
`migrate into damage sites [64,67]. However, repair processes are char-
`acteristic to the early disease phase [68]. As the disease progresses the
`new progenitor cells succumbed to the hostile conditions within the in-
`flamed lesions and self repair mechanism drastically decline. Promoting
`
`repair beyond its limited spontaneous extent is thus a major goal for MS
`therapy.
`Reduced myelin damages detected by scanning electron microsco-
`py (SEM) and immunohistochemistry in EAE-inflicted mice treated
`by delayed suppression therapeutic GA regimen suggested the induc-
`tion of repair processes [61]. In a recent study we further established
`the ability of GA to augment myelin repair, by applying transmission
`electron microscopy (TEM), that facilitates the visualization of newly
`myelinated axons, in mice inflicted by relapsing–remitting PLP-induced
`EAE, in which widespread demyelination is the main pathological man-
`ifestation [62]. Ultrastructural quantitative analysis of the relative
`remyelination compared to demyelination in the spinal cord of these
`mice provided evidence for significant augmentation of remyelination
`after GA treatment, by 7 and 3 fold over untreated mice, when treat-
`ment was applied during the first or the second disease exacerbation,
`respectively.
`The mode of action of GA in this system was attributed to increased
`proliferation, and survival of OPCs and their recruitment into injury
`sites, thus enhancing myelin repair in situ [56,61]. Furthermore, GA
`treatment induced a morphological transformation of OPCs from the
`earlier bipolar to the more mature multiprocessed form, suggesting its
`effect on the differentiation